The DNA damage response (DDR) senses and repairs genotoxic insults, arising from both endogenous metabolic processes and exogenous sources such as ultraviolet or cosmic radiation. Misrepair of these lesions results in mutations which lead to the development of cancer. Template damage, nucleotide imbalances, and difficult to replicate sequences cause replication stress and provide another potential source of mutations. The Replication Stress Response (RSR), an intrinsic cellular response to replication stress, functions during normal and stressed replication to stabilize stalled replication forks and promote the completion of DNA replication. The protein kinase ATR senses and responds to stalled replication forks, where it phosphorylates numerous substrates to begin RSR signaling. This proposal will use hydroxyurea (HU), which effects rapid replication fork stalling, to study th function of ATR using newly developed inhibitors. I hypothesize that ATR regulates stalled and collapsed replication forks to promote fork restart and avoid pathogenic fork intermediates. To test this hypothesis, I will utilize the newly developed iPOND methodology to probe replisome stability in response to HU and ATR inhibition. Furthermore, I will determine the involvement of several genetic pathways in modulating the severe HU sensitivity of ATR inhibited cells and in the generation of an unusual DNA structure where the newly synthesized DNA becomes single stranded. In this analysis, I will include the structure specific nuclease MUS81, which my preliminary data suggests functions upstream of nascent-strand ssDNA generation. Previous reports indicate that MUS81-deficient cells suffer from HU sensitivity, so this proposal will test the concept that checkpoint signaling regulates MUS81 activity at stalled forks. Finally, I will combine the iPOND methodology with the unbiased approach, mass spectrometry, to identify new RSR proteins. The recent literature includes several reports of new RSR factors. Given this rate of discovery, many new RSR factors likely remain undiscovered. I anticipate that this powerful approach will allow identification of new RSR proteins associated with newly synthesized DNA under normal, stressed, and ATR inhibited conditions. In summary, this proposal will provide a significant step forward in our understanding of how ATR functions in response to replication stress to mediate protein recruitment, replisome stabilization, and restart of replication.